The detection of closely related genetic variants is a significant challenge of analytical diagnostics. Pathogens such as, for example, viruses and bacteria, generally mutate frequently and form such genetic variants.
For example, the nucleic acid sequences of human immunodeficiency virus (HIV-1) having different origins, are different from each other. The different types of HIV-1 are divided into groups and subtypes. The major group M consists of ten currently identified subtypes, designated as subtypes A through H, J and K In addition to M-group viruses, two other groups, N and O, have been identified (Simon et al, 1998, Nature Med, 4:1032-1037). Within groups and subtypes, new strains of the virus are continuously being generated due to the error-prone nature of the HIV-1 replicative machinery.
Similarly, hepatitis C virus (HCV) does not exist as a homogeneous RNA population. Even within a single infected individual, numerous heterogeneous viral genomes (quasispecies) may co-exist. In addition, multiple genotypes of HCV have been identified on the basis of nucleotide sequence analysis of viral variants isolated from different geographic regions. There are currently six main HCV genotypes, classified numerically from 1 to 6. Genotypes are further subdivided according to subtype.
Due to this genetic variation of pathogens within a species, the range of diagnostic tests that provide reliable results are highly limited. Most detection methods currently available for detecting pathogens in a sample are based either on the detection of the pathogens' antigens, pathogen-induced antibodies, or the pathogens' intrinsic enzymes, e.g. intrinsic HIV reverse transcriptase. In addition to being inconvenient, such methods are frequently not very sensitive. For example, the method currently implemented by blood banks for screening of HIV-1 infection in blood donors is the detection of antibodies to virus proteins. This method fails to detect individuals in the early acute phase of the infection who have not yet developed diagnostic antibodies to the virus.
Screening methods that are based on the detection of nucleic acid sequences are sensitive and convenient. However, these tests may not always be reliable for detection of closely related genetic variants.
One of the currently available nucleic acid sequence-based detection methods utilizes molecular beacons (Tyagi and Kramer, 1996, Nat. Biotechnol., 14(3):303-308). Molecular beacons are single-stranded oligonucleotide probes that have a stem-loop structure. (See FIG. 1.) The loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm; and a quenching moiety is attached to the end of the other arm. The hybridization of the arms of the stem to each other keeps these two moieties in close proximity, causing the fluorescence of the fluorophore to be quenched by energy transfer (FIG. 1a). In the presence of the beacon's complementary DNA target, the loop structure hybridizes to the target, preventing the arms of the stem from remaining hybridized. The fluorophore and quencher are physically separated, and fluorescence is obtained (FIG. 1b).
Molecular beacons are currently used for real-time quantitative PCR. PCR primers are designed to amplify a specific segment of DNA, usually less than 200 base pairs in length. The beacon is typically designed so that its loop is complementary to a short (20-25 b.p.) region on one of the amplified DNA strands. The complementary region of these amplified DNA strands is the portion of these strands which has been added to the primers.
Molecular beacons are highly sequence-specific. In fact, one of the principle applications of this technology in recent years has been for allele discrimination or “molecular genotyping.” The sensitivity of molecular beacons to sequence variation permits discrimination between even single nucleotide polymorphisms in a give target sequence (Tyagi et al, 1998, Nat. Biotechnol., 16(1):49-53; Kostrikis et al., 1998, Science, 279:5354:1228-9; Marras et al., 1999, Genet. Anal., 14(5-6)151-6; Tapp et al., 2000, Biotechniques, 28(4):732-8).
To date, this sensitivity to sequence variation has severely limited the application of molecular beacon technology to the diagnosis of viral infection. The molecular beacons cannot efficiently detect the variant sequences of DNA or RNA targets. For example, a beacon designed to recognize PCR product from HIV strain A may not recognize PCR product from HIV strain B. (See FIG. 2.)
Thus, the present technology would require several different beacons to allow for the detection of all the different genotypes of the virus. That is, even though some highly conserved regions of the genome of HIV-1 are known to exist, it is likely that several different beacons would be needed to detect all the known subtypes of this virus. Moreover, even with the use of several different beacons, other variants of HIV-1 that have not been identified may not be detected.
Thus, current technology does not provide a convenient or efficient diagnostic assay for detection of all related genetic variants of pathogens.
There is an urgent need for sensitive, convenient nucleic acid-based screening assays capable of detecting closely related genetic variants. For example, there is a need for assays capable of detecting viruses, bacteria and other pathogens, directly in contaminated blood. Such assays are needed to detect blood or plasma units from individuals in the early acute stages of a pathogen infection, i.e., before the individual has developed diagnostic antibodies to the virus.
Accordingly, one of the purposes of the present invention is to overcome the above limitations in the prior art by providing a convenient and efficient diagnostic assay for detection of multiple variants of a particular target nucleic acid molecule.